LOCUS       sequence-321980-        7687 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for expression of the SpCas9 KG variant with sgRNA in E. 
            coli: KG(SpCas9, D1332K/R1333G) and UV5-sgRNA (hEGFP spacer).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7687)
  AUTHORS   Goldberg GW, Spencer JM, Giganti DO, Camellato BR, Agmon N, Ichikawa
            DM, Boeke JD, Noyes MB
  TITLE     Engineered dual selection for directed evolution of SpCas9 PAM 
            specificity.
  JOURNAL   Nat Commun. 2021 Jan 13;12(1):349. doi: 10.1038/s41467-020-20650-x.
  PUBMED    33441553
REFERENCE   2  (bases 1 to 7687)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-01-13"; volume: "12(1):349. doi"; pages: " 
            10.1038/s41467-020-20650-x"
COMMENT     Sequence Label: pGG422_UV5
FEATURES             Location/Qualifiers
     source          1..7687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(12..17,18..35,36..42)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    50..66
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     55..77
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             305..4411
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIKGKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     misc_RNA        4507..4582
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(4594..4618)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     rep_origin      4748..5203
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4835..4854)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5045..5066
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5229..5333
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5334..5402,5403..6194)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5552..5571)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6365..6953
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6854..6873
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7107..7124
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7139..7279
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7205..7224)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7365..7387
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7381..7572)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gaattccggg ctttacactt tatgcttccg gctcgtataa tgtgtcgact tgtgagcgga
       61 taacaatttc acacaggaaa cagctatgcc tcaacagcag caaatgcaac ctcccaattc
      121 aagtgcggac aacaaccctt tgcaacagca atcatcacaa aataccgtac caaacgtcct
      181 caaccaaatt aaccaaatct tttctccaga ggagcaacgc agcttattac aagaagccat
      241 cgaaacctgc aagaattttg aaaaaacaca attgtaagtc tcggctccaa gaaggagata
      301 taccatggat aagaaatact caataggctt agatatcggc acaaatagcg tcggatgggc
      361 ggtgatcact gatgaatata aggttccgtc taaaaagttc aaggttctgg gaaatacaga
      421 ccgccacagt atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc
      481 ggaagcgact cgtctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat
      541 ttgttatcta caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca
      601 tcgacttgaa gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt
      661 tggaaatata gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg
      721 aaaaaaattg gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc
      781 gcatatgatt aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag
      841 tgatgtggac aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa
      901 ccctattaac gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc
      961 aagacgatta gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg
     1021 gaatctcatt gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc
     1081 agaagatgct aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt
     1141 ggcgcaaatt ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc
     1201 tattttactt tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc
     1261 ttcaatgatt aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt
     1321 tcgacaacaa cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata
     1381 tgcaggttat attgatgggg gagcgagcca agaagaattt tataaattta tcaaaccaat
     1441 tttagaaaaa atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct
     1501 gcgcaagcaa cggacctttg acaacggctc tattccccat caaattcact tgggtgagct
     1561 gcacgctatt ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa
     1621 gattgaaaaa atcttgactt ttcgcatccc ttattatgtt ggtccattgg cgcgtggcaa
     1681 tagtcgtttt gcatggatga ctcggaagtc tgaagaaaca attaccccgt ggaattttga
     1741 agaagttgtc gataaaggtg cttcagctca atcatttatt gaacgcatga caaactttga
     1801 taaaaatctt ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac
     1861 ggtttataac gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt
     1921 tctttcaggt gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt
     1981 aaccgttaag caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga
     2041 aatttcagga gttgaagata gatttaatgc ttcattaggg acataccatg atttgctaaa
     2101 aattattaaa gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat
     2161 tgttttaaca ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata
     2221 tgctcacctc tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg
     2281 gggacgtttg tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat
     2341 attagatttt ttgaaatcag atggttttgc caatcgcaat tttatgcagc tgatccatga
     2401 tgatagtttg acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag
     2461 tttacatgaa catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca
     2521 gactgtaaaa gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat
     2581 cgttattgaa atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga
     2641 gcgtatgaaa cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca
     2701 tcctgttgaa aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg
     2761 aagagacatg tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga
     2821 tcacattgtt ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg
     2881 ttctgataaa aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat
     2941 gaaaaactat tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa
     3001 tttaacgaaa gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg
     3061 ccaattggtt gaaactcgcc aaatcactaa gcatgtggca caaattttgg atagtcgcat
     3121 gaatactaaa tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa
     3181 atctaaatta gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa
     3241 caattaccat catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa
     3301 gaaatatcca aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg
     3361 taaaatgatt gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta
     3421 ctctaatatc atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa
     3481 gcggccgcta atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga
     3541 ttttgccaca gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga
     3601 agtacagacc ggtggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct
     3661 tattgctcgt aaaaaagact gggacccaaa aaaatatggt ggttttgata gtccaacggt
     3721 agcttattca gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc
     3781 cgttaaagag ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat
     3841 tgacttttta gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc
     3901 taaatatagt ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga
     3961 attacaaaaa ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc
     4021 tagtcattat gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt
     4081 ggagcagcat aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg
     4141 tgttatttta gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga
     4201 caaaccaata cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg
     4261 agcgcccgct gcgttcaagt acttcgacac cacgatcaag gggaagaggt acacgtctac
     4321 aaaagaagtt ctagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg
     4381 cattgatttg agtcagctag gaggtgactg atagggatcc ggtacctaat agtgagtcga
     4441 gctgcccggg ttgacacgca agctcagtcc taggtataat actagtccca tcctggtcga
     4501 gctggagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     4561 aaaagtggca ccgagtcggt gctttttttc tacacccagc tttcttgtac aaagttggca
     4621 ttaactcgag gctagagact agaaaaaggc cgacaagtcc cgctccgctg aagatcctgg
     4681 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     4741 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4801 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4861 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4921 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     4981 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5041 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     5101 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     5161 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     5221 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5281 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5341 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5401 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5461 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5521 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5581 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5641 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5701 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5761 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5821 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5881 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5941 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6001 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggt tctcgcggta
     6061 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6121 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6181 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6241 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6301 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6361 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6421 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6481 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     6541 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6601 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6661 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6721 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6781 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6841 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6901 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6961 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7021 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7081 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     7141 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     7201 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     7261 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     7321 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     7381 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     7441 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     7501 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     7561 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     7621 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     7681 ggaacgg
//
